Formulations and method for isolating nucleic acids from optional complex starting material and subsequent complex gene analytics

ABSTRACT

The subject of the invention are formulations not containing chaotropic components for isolating nucleic acids with binding to a solid phase, in particular of DNA, from optional complex starting materials and quantities containing a lysis/binding buffer system which comprises at least one antichaotropic salt component, a solid phase and wash and elution buffers known as such. The lysis/binding buffer system may be an aqueous solution or a solid formulation in reaction vessels ready for use. All carriers used for isolation by means of chaotropic reagents, preferably glass fiber mats, glass membranes, silica carriers, ceramics, zeolites or materials showing negatively functionalised surfaces or chemically modified surfaces which may be converted to a negative charge potential may serve as a solid phase.  
     Furthermore, the subject of the invention is a method for isolating nucleic acids, in particular DNA, from optional complex starting materials with using the formulations according to the invention which is characterized by the lysis of the starting material, binding of nucleic acids to a carrier, washing of the nucleic acids bound to the carrier and elution of nucleic acids

[0001] Subject of the invention are formulations which do not containchaotropic components for isolating nucleic acids with binding them to asolid phase, in particular DNA, from optional complex starting materialsand quantities which contain a lysisibinding buffer system involving atleast one antichaotropic salt component, a solid phase and wash andelution buffers known as such. The lysisibinding buffer system may bepresent as an aqueous solution or as a solid formulation of reactionvessels ready for use. All carriers applied for isolation by means ofchaotropic reagents, preferably glass fiber mats, glass membranes,silicon carriers, ceramics, zeolites or materials possessing negativelyfunctionalised or chemically modified surfaces which may be converted toa negative charge potential, may serve as a solid phase.

[0002] Furthermore, the subject of the invention is a method forisolating nucleic acids, in particular DNA, from optional complexstarting materials with using the formulations according to theinvention which is characterized by the lysis of the starting material,binding of nucleic acids to a carrier, washing of the nucleic acidsbound to the carrier and elution of the nucleic acids, with amplifyingselected sequence sections subsequently, if necessary, and analyzing themultiplied gene sections in the very same reaction cavity subsequently,if necessary. Fields of application of the methods are all laboratoriesdealing with DNA isolation such as forensic medicine, food diagnostics,medical diagnostics, molecular biology, biochemistry, geneticengineering and all other adjacent fields.

[0003] Given classical conditions, DNA are isolated from cells andtissues by decomposing the starting materials which contain nucleicacids under strongly denaturating and reducing conditions, with partlyalso using protein-degrading enzymes, purifying the escaping nucleicacid fractions in phenol/chloroform extraction steps and obtainingnucleic acids by means of dialysis or ethanol precipitation from theaqueous phase (Sambrook, J.; Fritsch, E. F.; and Maniatis, T., 1989,CSH, “Molecular Cloning”).

[0004] The “classical methods” for isolating nucleic acids from cellsand, in particular, from tissues, are very time-consuming (partly morethan 48 hours), require a remarkable expenditure on apparatuses and,apart from that, are not implementable under field conditions. Inaddition, such methods are due to the used chemicals phenol andchloroform, to a not insignificant degree, dangerous to health.

[0005] Various alternative methods for isolating nucleic acids fromvarious biological starting materials allow to avoid the expensive andunhealthy phenol/chloroform extraction of nucleic acids and a reductionof the expenditure of time.

[0006] All these methods are based on a method for the preparative andanalytical purification of DNA fragments from agarose gels developed byVogelstein and Gillespie (Proc. Natl. Acad. Sc. USA, 1979, 76, 615-619).The method combines the dissolution of agarose containing the DNA bandsto be isolated in a saturated solution of a chaotropic salt (NaJ) withbinding DNA to glass particles. The DNA fixed on the glass particles issubsequently washed with a wash solution (20 mM of tris HCl [pH 7.2];200 mM of NaCl; 2 mM of EDTA; 50% v/v ethanol) and thereupon dissolvedfrom the carrier particles.

[0007] Till the present day this method has been a few times modifiedand is, for the time being, applied for various processes of extractionand purification of nucleic acids of various origin (Marko, M. A.;Chipperfield, R. and Bimboim, H. G.; 1982, Anal. Biochem., 121,382-387).

[0008] In addition, today exists worldwide also a multitude of reagentsystems primarily for purifying DNA fragments of agarose gels and forisolating plasmide DNA from bacterial lysates, yet also for isolatingnucleic acids with longer chains (genomic DNA, cellular total RNA) fromblood, tissue or also cell cultures.

[0009] All these commercially available kits are based on the principleof binding nucleic acids to mineral carrier in the presence of solutionsof various chaotropic salts, which is sufficiently known, and usesuspensions of finely grinded glass powder as carriers (e.g. glass milk,BIO 101, La Jolla, Calif.), diatom earths (company Sigma) or also silicagels (Diagen, DE 41 39 664 A1).

[0010] A method for isolating nucleic acids practicable for a multitudeof various uses is represented in U.S. Pat. No. 5.234.809 (Boom). There,a method for isolating nucleic acids from starting materials whichcontain nucleic acids by incubating the starting materials with achaotropic buffer and a solid phase binding DNA is described. Thechaotropic buffers implement the lysis of the starting materials as wellas binding of nucleic acids to the solid phase. The method is wellsuited for isolating nucleic acids from small quantities of materials,being in practice especially applied in the field of isolating viralnucleic acids.

[0011] Specific modifications of these methods relate to the use ofnovel carriers which show advantages in application as to specificaspects (Invitek GmbH WO-A 95/34569).

[0012] Yet, decisive drawbacks of the method of isolating nucleic acidsfrom complex starting materials on the basis of incubating the startingmaterial with a chaotropic buffer and a solid phase consist a .o. in thefact that the decomposition of cells to be brought about by thechaotropic buffers is not applicable to all materials or works onlyextremely insufficiently and with a high expenditure of time also forbigger quantities of starting materials. Apart from that, mechanichomogenisation methods are required if e.g. DNA has to be isolated fromtissue samples. Furthermore, various concentrations of differentchaotropic buffers have to be always used for investigating variousaspects. Thus, the method is, in no way, universally applicable.

[0013] Though problems arising due to a possibly difficult lysis of thestarting material may be solved by a number of commercially availableproducts for isolating nucleic acids (especially for isolating genomicDNA from complex starting materials), they have the big drawback that nolonger a classical “single tube” method is concerned which characterizesthe method according to the U.S. patent as the lysis of the startingmaterial is carried out in a usual buffer including a proteolyticenzyme. The chaotropic ions required for the subsequent binding ofnucleic acids e.g. to centrifugation membranes have to be addedseparately to the lysis batch after completing the lysis. But theycannot form part of the lysis buffer as the protein destroying functionof chaotropic salts is known and would, of course, immediately destroythe proteolytic enzyme required for an efficient lysis.

[0014] That is why the methods of isolating nucleic acids with usingchaotropic salts have gained acceptance worldwide in spite of a numberof drawbacks and are used a million times by means of commerciallyavailable products. These systems are extremely simple to apply andfollow always the principle of lysis of starting materials, thesubsequent binding of nucleic acid to the solid phase of a glass orsilica membrane which is in a centrifugation column on a carriersuspension, washing of the bound nucleic acids and the subsequentelution of the nucleic acids with a buffer of an insignificant ionicstrength.

[0015] All these systems are based on binding the nucleic acids to therespective carrier surfaces in the presence of chaotropic salts, i.e. atleast one buffer solution contains a chaotropic salt as main component.This refers possibly already to the lysis buffer or in the case ofsystems including proteolytic enzymes a required binding buffer which isadded after completing the lysis of the starting material.

[0016] The series of Hofneister for salting out negatively charged,neutral or basic protein solutions form the basis for chaotropic salts.Thus, chaotropic salts are characterized by denaturing proteins,increasing the solubility of nonpolar substances in water and destroyinghydrophobic interactions. According to the state of the art notablythese properties, also with buffer systems of chaotropic salts, destroythe superior structure of the aqueous medium to thus bring about thebinding of the nucleic acids to selected solid phases. The mostimportant agents for isolating nucleic acid are sodium perchlorate,sodium iodide, potassium iodide, guanidine thiocyanate and guanidinehydrochloride. Yet, on the one hand, they are expensive and, on theother hand, and partly toxic or corrosive.

[0017] Till the present day a very great number of patent applicationsand granted patents have been based on this state of the art, wherealways variants of the method are concerned such as e.g. the use of newcarriers or more efficient wash buffers etc. with the basic principlebeing always the use of chaotropic salts for binding to a solid phasewhich consists of silica materials.

[0018] The physical-chemical principle of binding nucleic acids tomineral carriers in the presence of chaotropic salts is regarded to beacknowledged among international experts. Binding of nucleic acids tothe surfaces of the mineral carrier consists in the disturbance ofsuperior structures of the aqueous medium through which nucleic acidsare adsorbed to the surface of mineral materials, in particular glass orsilica particles. Disturbing the superior structures of the aqueousmedium requires always the presence of chaotropic ions. In case of highconcentrations of chaotropic salts the reaction proceeds nearlyquantitatively. Owing to these physical-chemical findings describedexperts proceed on the fact that all commercially available systems forisolating nucleic acids have to contain buffer compositions with a highionic strength of the chaotropic salts for binding nucleic acids to asolid phase binding nucleic acids.

[0019] The more surprising were the findings according to the inventionthat formulations containing antichaotropic salts in a lysis/bindingbuffer system are equally and better suited for isolating nucleic acidsfrom optional, in particular complex starting materials.

[0020] The invention is implemented according to the claims. That is whythe subject of the invention are formulations and methods which do notcontain chaotropic components for isolating nucleic acids with bindingthem to a solid phase, in particular DNA, from optional complex startingmaterials which contain a lysis/binding buffer system involving at leastone antichaotropic salt component, a solid phase and wash and elutionbuffers known as such. Antichaotropic components in the sense of theinvention are ammonium, caesium, sodium and/or potassium salts,preferably ammonium chloride.

[0021] In addition, the lysisibuffer system contains detergents known assuch and additives such as e.g. tris-HCL, EDTA, polyvinyl pyrrolidone,CTAB, triton X-100, N-lauryl sarcosine, sodium citrate, DTT, SDS and/orTween. In a preferred variant of execution the lysis/binding buffersystem contains an alcohol such as e.g. ethanol and isopropanol andenzymes, if necessary, preferably enzymes degrading protein, e.g.proteinase, for binding to the solid phase. With the invention theprinciple corresponding to the state of the art may be used to solve aspecific problem of isolating nucleic acid or to optimize and makeefficient an existing variant as regards specific relevant parameters.Thus it is suited for being applied as a fully automatic high-throughputmethod.

[0022] Completely unexpected and unlike the known state of the art,nucleic acids, in particular genomic DNA, may be bound according to thepresent invention to a mineral supporting agent with lysis/bindingbuffer systems not containing chaotropic salt components and also elutedunder the usual conditions.

[0023] Furthermore it was stated that a multitude of completelydifferent salts as components of lysis/binding buffer systems also usualas such, if necessary, are sufficient for binding nucleic acids toclassical carriers on the basis of glass or silica.

[0024] To our particular surprise, the best results may be achieved withsalts showing after their chemical-physical characteristics theabsolutely opposite effects with regard to the chaotropic salts used sofar for binding nucleic acids. Thus, we may call these saltsantichaotropic.

[0025] Thus, it was possible to achieve at least the same quantitativeand qualitative results in extractions of genomic DNA from variouscomplex starting materials (e.g. blood, tissue, plants) withlysis/binding buffers the main component of which were e.g. ammoniumsalts instead of chaotropic salts (commercial extraction kits), keepingthe other reaction components, carriers usual so far constant and givencompletely the same reaction course.

[0026] Thereby, notably the ammonium ion is the ion in the Hofmeisterseries showing in chemical-physical respect properties which areabsolutely opposite to the known chaotropic ions of this series.

[0027] Solely by replacing the chaotropic salt component so far used byan antichaotropic salt component in the lysis/binding buffer, with allother parameters being constant, on the surfaces of the solid carriersknown as such an at least adequate quantitative isolation of nucleicacids is possible.

[0028] That means that it is in the same way possible to isolate nucleicacids also from complex starting materials by a salt not denaturatingproteins but stabilizing them which does not increase the solubility ofnonpolar substances in water but reduces it and which does not destroyhydrophobic interactions but intensifies them, to purify and supply themto applications usual as such.

[0029] With the present invention a novel, alternative mechanism forbinding nucleic acids to solid, preferably mineral carriers, and on thisbasis, a universally applicable novel method for isolating nucleic acidsfrom complex starting materials is provided.

[0030] Thus, the invention allows to use an alternative chemism asessential component of the respective test kits (formulations) throughthe use of novel compositions of lysis/binding buffers on the basis ofchaotropic salts for isolating nucleic acids, especially for isolatinggenomic DNA based on binding the nucleic acids to the various solidphases of silica or glass materials usual as such.

[0031] Thereby, the method according to the invention usingantichaotropic salts follows the process courses for isolating nucleicacid known from the laboratory routine practice and is characterized by:

[0032] 1. Lysis of starting material

[0033] 2. Binding of nucleic acids to a solid phase (centrifugationcolumn or suspension)

[0034] 3. Washing of the bound nucleic acids

[0035] 4. Elution of the nucleic acids with a low-salt buffer known assuch.

[0036] The invention allows a highly efficient and fast isolation ofnucleic acids, especially genomic DNA from an optional and, ifnecessary, complex starting material. The antichaotropic ions requiredfor binding may be a component of the lysis/binding buffer even whenincluding proteolytic enzymes. The method according to the invention isthus easily and universally applicable.

[0037] The isolation of nucleic acids, in particular DNA, from optionalstarting materials is effected by incubation of the starting materialscontaining the nucleic acid without using chaotropic substances whichare brought into contact with

[0038] the lysis/binding buffer system which comprises an aqueoussolution containing an antichaotropic salt component, at least adetergent, if necessary, additives and, if necessary, an enzyme,

[0039] and an optional solid phase, preferably glass fiber mats, glassmembranes, glasses, zeolites, ceramics and other silica carriers

[0040] thus bringing about the lysis of the starting material and thesubsequent binding of DNA to the solid phase. Subsequently, the boundnucleic acid is washed according to methods known as such and dissolvedfrom the solid phase.

[0041] In case of special extraction protocols an additional detergent,an alcohol or a detergent/alcohol mixture may be added to the lysisbatch, if necessary.

[0042] Preferred starting materials are compact plant materials such ase.g. fruit, seeds, leaves, needles etc., clinically relevant samplessuch as whole blood, tissue, microbioptates, paraffin-coated materials,ercp-samples, swabs, foodstuffs such as e.g. fish, sausage, tins, milk,forensic samples such as e.g. hair roots, cigarette butts, blood stainsand other samples containing DNA.

[0043] Preferred ions in the sense of the invention are theantichaotropic ammonium ions represented in the Hofmeister series,caesium ions and potassium and sodium ions or combinations of theseions, preferably ammonium chloride. They are used in an ionic strengthof 0.1 to 8 M for lysis/binding.

[0044] For binding nucleic acids, in particular DNA, to the solidcarriers already low concentrations of these salts of preferably ≦1 Mwill be sufficient, in certain applications preferably evenconcentrations of ≦0.5 M with higher ionic concentrations beingsuccessful in the quantitative isolation of nucleic acids from biggerquantities of starting materials.

[0045] By using antichaotropic salts which have a protein stabilizingeffect as essential components of a lysis buffer in a preferred form ofexecution of the invention also proteolytic enzymes such as e.g.proteinase K may be added for supporting and making the lysis processmore effective, thus also antichaotropic salts of a high ionic strengthas e.g. 5 M are added for the required decomposition of cells, thusallowing a quantitative isolation of nucleic acids.

[0046] Buffer systems of the state of the art with the known chaotropicsalts may not contain proteolytic enzymes of the required high ionicstrength as it is, in general, necessary for a quantitative isolation ofnucleic acids. Thus, they have always to be subsequently added forbinding nucleic acids to the solid phases.

[0047] In the lysis buffers/binding buffers according to the inventionpreferably anionic, cationic or neutral proteolytic enzymes such as e.g.SDS, triton X-100, Tween or CTAB are used as detergents.

[0048] After completing the lysis of the starting material thesuspension is separated from the components not yet completely lysed ina short centrifugation step, if necessary, and directly incubated withthe material binding DNA or, as already described, after adding anadditional detergent, alcohol or a detergent/alcohol mixture incubatedwith the solid phase. In the lysis buffer system there are possiblyadditional insignificant concentrations (<50 mM) of EDTA and/ortris-HCl. For isolating DNA from very strongly polluted startingmaterials preferably also 2-4% polyvinyl pyrrolidone or other knownsubstances are added to the buffer system to bind inhibitory componentsselectively.

[0049] For instance commercially available glass fiber mats incentrifugation columns, silicone compounds such as SiO₂ of a variousparticle size have proved to be remarkable as binding material for theDNA to be isolated. Thus, all the materials used for isolating nucleicacids by means of chaotropic buffers may be used.

[0050] After incubation with the DNA binding material the lysate isseparated from the binding material by a short centrifugation step.Subsequently, it is washed with a wash buffer e.g. consisting of atleast 50% ethanol and a low salt concentration, if necessary, e.g. NaCl,in a way known as such, the carrier is dried and the bound DNA is elutedby means of a low salt buffer (tris-HCl; TE; water) known as such and ata preferable temperature of 50-70° C.

[0051] A further variant of applying the invention consists in addingproteolytic enzymes, preferably proteinases such as e.g. proteinase K,for the lysis of starting materials which are difficult to decompose,e.g. compact tissue samples, hair roots or for optimizing the efficiencyof lysis and reducing the required lysis periods.

[0052] Thus, the invention allows to apply universally applicablemethods of isolating nucleic acids, in particular DNA, from all startingmaterials containing DNA as well as from optional quantities of mostvarious starting materials in novel combinations of antichaotropic saltsas essential components of lysis buffer mixtures, with all carriers sofar used and their variants being equally efficiently used and theregulations governing the isolation of nucleic acids which have been sofar applied being identically usable.

[0053] In its most general variant of application by means of the methodaccording to the invention an extraction of nucleic acid from allselected complex starting materials corresponding to the state of theart, i.e. by means of the new universal buffer system, the highlyefficient lysis and subsequent binding of nucleic acid to a mineralcarrier from compact plant materials (e.g. fruit, seeds, leaves, needlesetc.), from clinically relevant samples (e.g. whole blood, tissue,microbioptate, paraffin-coated materials, ercp-samples, swabs), fromfoodstuffs (e.g. fish, sausage, tins, milk), from forensic samples (e.g.hair roots, cigarette butts, blood stains) as well as from otherstarting materials may be carried out successfully, extremely easiliyand very quickly.

[0054] A further advantage of the method consists in the fact that itallows to isolate DNA highly efficiently from an extremely smallquantity of starting materials (e.g. isolation of DNA from 1 μl of wholeblood, hair roots, microbiopsy<1 mg) as well as from very big quantitiesof starting materials such as e.g. 50 ml of whole blood, 1 g of tissuematerial, <1 g of plant material.

[0055] So Further advantages of replacing chaotropic salts byantichaotropic salts consist in the fact that the buffers used have nolonger toxic or corrosive effects owing to chaotropic chemicals lacking.

[0056] Apart from a most general variant of execution optimizations ofthe extraction method related to specific applications allow to isolatenearly quantitatively the DNA quantities contained in the startingsample. It is astonishing that higher DNA yields may be obtained bymeans of the method according to the invention without using chaotropicions of a high concentration binding DNA according to the state of theart than this has been possible so far by means of commerciallyavailable and highly optimated extraction kits.

[0057] A selection of the respective comparative results achieved bymeans of commercially available extraction kits is represented in theexamples of execution. These results demonstrate clearly the potentialsof the invention.

[0058] Apart from isolating DNA from all complex starting materialswhich contain DNA a further variant of applying the method according tothe invention allows to isolate highly efficiently plasmide DNA frombacterial lysates and without using chaotropic salts as such requiredfor binding plasmide DNA to mineral carriers according to the state ofthe art. Thus, in accordance with the process steps of isolatingplasmide DNA by means of basic lysis known to the expert the requiredso-called neutralization reaction is carried out by means of theclassical solution III (Maniatis and Sambroek) and this solution III ina thus existing dual function implements also simultaneously the bindingof the plasmide DNA to the solid carriers customary as such. Thus, theusual addition of a chaotropic guanidinium hydrochloride is not requiredfor binding the plasmide DNA.

[0059] The bound plasmide DNA is also washed in a way known as such andeluted from the carrier. The method is suited for isolating plasmide DNAfrom all starting quantities used (mini to giga). Thereby, the plasmideDNA yields obtained are identical with the yields isolated according totraditional, commercially available methods. However, the methodaccording to the invention is much more moderate in price than all othersystems known as chaotropic salts are very cost-intensive.

[0060] Thus, the method using antichaotropic salts is excellently suitedfor designing automatable systems for isolating plasmide where theprice/preparations are a decisive criterion of selection, as is known.

[0061] The formulations according to invention allow in a surprising waythe access to further, highly interesting and novel applications in thefield of isolating nucleic acids and of diagnostics.

[0062] In a further form of applying the invention the present newlysis/binding buffer systems containing at least one antichaotropic saltcomponent are in a position to bind nucleic acids to solid phases whichhave a negatively charged surface or surfaces which have a negativecharge potential.

[0063] Methods and means of purifying nucleic acids with the binding ofnucleic acid being effected to chemically modified solid phases areknown from the state of the art (U.S. Pat. No. 5,523,392; Purificationof DNA on Aluminium Silicates and Phosphosilicates; U.S. Pat. No.5.503.816; Silicate Compounds for DNA Purification; U.S. Pat. No.5,674,997; DNA purification on modified Silicates; U.S. Pat. No.5,438,127; DNA Purification by solid phase extraction using a PCl₃modified glass fiber membrane; U.S. Pat. No. 5,606,046: DNA purificationby solid phase extraction using trifluorimetric acid washed glass fibermembrane; United States Patent: DNA purification by solid phaseextraction using glass fiber membrane previously treated withtrifluoroacetic acid and then with fluoride ion, hydroxide ion, or BCl₃,U.S. Pat. No. 5,610,291: Glass fiber membranes modified by treatmentwith SiCl₃, AlCl₃ or BCl₃ and washing with NaOH to set as a DNAadsorbant; U.S. Pat. No. 5,616,701: DNA purification by solid phaseextraction using hydroxide-washed glass fiber membrane; U.S. Pat. No.5.650.506: Modified glass fiber membranes useful for DNA purification bysolid phase extraction).

[0064] Thereby, the prerequisite for this binding of nucleic acid isalways that the membranes used for the binding are doped with positiveionic charges by chemical modification reactions. Thus, it is obviousthat a binding will be brought about by a Coulomb interaction betweenthe positively charged surface of the membranes used and the negativeionic charge of the phosphate bone of nucleic acids. Thus, the principleof binding nucleic acids to positively charged solid phases already formany years has been a standard application, e.g. for DNA/RNA blottingtechniques on positively charged nylon filters, as is known, which issufficiently known among experts.

[0065] Yet, a completely essential drawback of this method described isthat it is not suited for isolating nucleic acids, i.e. it is completelyimpossible to isolate nucleic acids from complex starting materials.Starting material are always already isolated nucleic acids which haveto be isolated in the way known as such as has been shown in the U.S.patent specifications cited. In particular, one aspect does not yet seemto be clear to the expert, thereby. The binding conditions (bindingunder physiological buffer conditions) described and the elutionconditions are identical. There is not to be seen how the nucleic acidsmay be again dissolved from the membrane given the same bufferconditions for binding the nucleic acids to the positively chargedmembrane.

[0066] Finally, the represented means and the respective method may bepractically applied only in a very narrow way. Binding of syntheticallyproduced oligonucleotides to positive surfaces is also known. This is,on its turn, effected by utilizing the Coulomb interactions, i.e. on thebasis of linking positive and negative charges, e.g. through modifiedoligonucleotides (linkage with amino linkers or phosphate linkers).These variants do not allow either an isolation of nucleic acids fromcomplex starting materials.

[0067] As has been explained in detail there exist alternative forms ofbinding nucleic acid for punrying membranes with a sufficient positivecharge which do not represent methods for isolating nucleic acids.Binding of nucleic acids is brought about by Coulomb forces based on theinteractions between positive ionic charges of the membranes andnegative ionic charges of the nucleic acid bone. Thus, this principleseems to be logically explainable.

[0068] Based on the isolation of nucleic acids from complex startingmaterials with antichaotropic salts according to the invention asurprising phenomenon was detected. Thus, it became evident that alsonegatively charged surfaces or surfaces which may be converted to anegative charge potential are suited for binding nucleic acids using thelysis/binding buffer systems according to the invention. In general, wecould not expect such a possibility as not a binding but a repulsionwould have to occur owing to equal charge potentials.

[0069] The negatively functionalised surfaces or surfaces withpotentially negative modifications according to the invention areproduced according to methods known as such. The photochemical couplingof an acetyl group, carboxyl group or hyxdroxyl group with the surfaceof a reaction vessel has e.g. proved to be suited.

[0070] The present variant opens up completely new prospects for acomplex nucleic acid analytics. Namely, there became obvious that thenucleic acid need not be already isolated for binding to negative orpotentially negative surfaces as in all variants described so far.Binding will be effected from the lysis reaction batch, i.e. thestarting sample containing the nucleic acid will be lysed and thereleased nucleic acids will be bound to the negatively charged surface(e.g. a microtest plate cavity or an Eppendorf reaction vessel).

[0071] The variant according to the invention allows now to applycompletely novel “single tube” and one step methods for isolatingnucleic acids from complex starting materials. Such methods provideimmense advantages in their application spectrum for users (simplicity,cheapness, reduction of waste, fastness, suitability for routine use,automatability etc.)

[0072] In addition, a further application of this variant is not only toextract nucleic acids in a reaction cavity but to allow also asubsequent target amplification and subsequent analysing in the samereaction vessel, if necessary, hybridisation reactions, if necessary, orsequencing on solid phases.

[0073] On this basis e.g. an 0.5 ml Eppendorf PCR vessel is modified bymeans of techniques known to experts by a negatively charged orpotentially negative functional group. The photochemical coupling of anacetyl group, carboxyl group or hydroxyl group with the surface of thereaction vessel is e.g. suited for that. Then, the sample selected forisolating nucleic acids (e.g. whole blood) is put into the reactionvessel and incubated with a lysis buffer containing the antichaotropicsalt fraction, with adding e.g. ammonium chloride, a detergent and aproteolytic enzyme and the vessel is incubated at 70° C. for 5 min.

[0074] To maximize the binding of nucleic acids a detergent/alcoholmixture may be still pipetted after completing the lysis of the startingmaterial. Then, the batch is shortly incubated and subsequently pouredoff the reaction vessel. Now, the nucleic acid is bound to thefunctionalised surface of the reaction vessel and is subsequentlyshortly washed with an alcoholic wash buffer and the alcohol is removedby incubation,e.g. at 70° C. Further, the elution of the bound nucleicacids is effected in an expert way by adding a low salt buffer (e.g. 10mM of tris-HCl) to the reaction vessel and a short incubation (e.g. 2min.) at e.g. 70° C. Thus, the nucleic acid is available for subsequentuse.

[0075] As has been shown, all reactions of isolating nucleic acid from acomplex starting material proceed in one reaction vessel, i.e. the lysisof the starting material, binding of nucleic acids, washing of the boundnucleic acids and elution of the nucleic acids, are implemented in andwith one reaction vessel.

[0076] The extraction kits of the Qiagen company at present mostfrequently applied worldwide need for lysing, binding, washing andelution always one filter cartridge and at least 4 separate reactionvessels, including, in addition, multiple centrifugation steps.

[0077] Contrary to this, the variant according to the invention allowsto extract nucleic acid without a single centrifugation step. Anenormous advantage relating to time may be also derived from this. Theseadvantages relate also to the methods of nucleic acid extraction citedin U.S. Pat. No. 5,234,809 described by Boom.

[0078] But apart from the potential extraction of nucleic acid the boundnucleic acid may also remain at the surface of the described 0.5 mlreaction vessel and be e.g. subsequently directly used for a PCRapplication by adding a complete PCR mix (primer, nucleotide, polymerasebuffer, Taq polymerase, magnesium), i.e. extraction and amplificationproceed then in the same reaction vessel.

[0079] These examples illustrate the enormous advantages and the wideapplicability derivable from the invention. In one variant it enablesthe whole process of preparation of samples via amplification and alsoanalysing, if necessary, to proceed e.g. in one reaction cavity. Thus,with the provision of modified reaction vessels (or also other solidsurfaces) and the appropriate lysis/binding buffers new standards aredeveloped in laboratories dealing with molecular biology and primarilywith nucleic acid diagnosing with the problems of contaminating sampleswhich are sufficiently known being drastically reduced by the newpotential solutions of application.

[0080] A further advantage and also a further application is that thenucleic acids fixed on the surface will remain stably fixed also atleast for a longer time, thus being available for a later treatment,i.e. the PCR will not have to follow immediately after extraction. Afurther field of application is the fully automated extraction ofnucleic acid and analysing, if necessary, using the surfaces bearingnegative or potentially negative charges which are described here(preferably plastic surfaces of suitable reaction cavities, e.g.microtest plates).

[0081] The lysisibinding buffer systems according to the invention withantichaotropic salts as main components including a proteolytic enzyme,if necessary, may be also provided as a solid formulation. For thispurpose the mixtures of salts and detergents, additives and enzymes, ifnecessary, will be aliquoted in usual reaction vessels and incubated fora few hours at 95° C. or lyophilysed according to methods known as suchand thus converted into a solid formulation.

[0082] These solid formulations in complex ready reaction mixtures forisolating nucleic acids are stable in long-time storage, i.e. also thebiological activity of the proteolytic enzyme component is maintainedduring long-time storage (see example of execution). Thereby, the stableformulation of lysis buffer mixtures is prepared without addingprotective additives known as such, simply by a low-temperaturelyophilisation.

[0083] All test kits for the extraction of nucleic acids offeredcommercially contain the required components individually, specificsolutions have to be prepared only by the user. Apart from that, thestability of the solutions is restricted. A further drawback consists inthe fact that the user has to consider multiple pipetting steps forvarious individual solutions while isolating nucleic acids using testkits customary at present. This increases the risk of contaminationdrastically, notably in the field of medical diagnostics. Furthermore,it is disadvantagous that e.g. owing to the limits of loading widelycustomary centrifugation columns which are mainly applied for isolatingnucleic acids also the quantity of the starting material is stronglylimited. This is due to the fact that lysis and binding buffers requiredfor the extraction have still to be added to the starting material.

[0084] By providing a stable formulation as a lysis mix stable instorage on the basis of antichaotropic salts the existing problems aresolved in a completely simple way. This formulation has the followingadvantages:

[0085] 1. Long-time storage of lysis buffer mixes ready for use.

[0086] 2. Stabilization of proteolytic enzymes in ready lysis mixturesand their long-time storage

[0087] 3. Use of bigger quantities of starting materials at an equaldimensioning of existing centrifugation columns (e.g. triplication ofthe starting quantity)

[0088] 4. Reduction of contamination risks by reducing pipetting stepsand solutions

[0089] 5. Uptake of sample in the ready lysis mix also outside thelaboratory and its long-time storage, if necessary

[0090] 6. Stable dispatch of samples and cooling.

[0091] The ready, solid, stable lysis buffer mixes consisting of amultitude of individual components including proteolytic enzymes, ifnecessary, are easily to handle (also by persons who do not have specialknowledge) as the reaction is started simply by adding a samplecontaining the nucleic acid to be isolated. Apart from that, we canproceed on the fact that the mixtures have a life time of at least 6months according to the substances they contain, thus a transport of thesample at room temperature is no longer a problem either.

[0092] The advantage of the solid formulations is based on the fact thatthe sample containing nucleic acids will be only put into the reactionvessel containing the lysis buffer stable in storage for the lysis ofthe nucleic acids contained in the sample materials and the sample islysed in the respective reaction vessel, if necessary, by adding water.Costly multiple pipetting steps which burden contamination arecompletely dropped. The problems known which are connected withcollecting and preparing clinical and forensic samples, given fieldconditions, are solved by the formulation according to the invention anda formulation easily to handle is available.

[0093] To our surprise, the practical application showed also that afteradding the starting material to be lysed, if necessary, when adding asolid sample after adding H₂O the solid formulation may be againconverted to a liquid phase without causing problems, given standardreaction conditions.

[0094] To sum up there is to be stated:

[0095] The subject of the invention is the use of antichaotropic saltsin formulations which do not contain chaotropic components for isolatingnucleic acids with binding to a solid phase, in particular DNA ofoptional complex starting materials. The formulations containlysis/binding buffer systems which have at least one antichaotropic saltcomponent, a solid phase and wash and elution buffers known as such.

[0096] The lysis/binding buffer system may be available as an aqueoussolution or as a solid formulation in reaction vessels ready for use.

[0097] All carriers may serve as solid phase used for isolatingchaotropic reagents, preferably glass fiber mats, glass membranes,silicon carriers and aerosiles or carriers which have a negativelycharged surface or chemically modified surfaces showing a negativecharge potential.

[0098] Furthermore, the subject of the invention is a method forisolating nucleic acids, in particular DNA, from optional complexstarting materials, using the formulations mentioned which ischaracterized by a lysis of the starting material, binding nucleic acidsto a carrier, washing the nucleic acids bound to the carrier and elutionof the nucleic acids.

[0099] Owing to the DNA quality achieved it is also well suited for thepreparative isolation and purification of DNA for use in gene therapy.

[0100] The subject of the invention are also solid formulations of lysisbuffer systems stable in storage and ready for use for isolating nucleicacid on the basis of antichaotropic salts available as mixes ready foruse in conventional reaction vessels. The solid formulations of thelysis buffer batches are activated by adding only the sample (in thecase of liquid samples such as e.g. whole blood, saliva, cellsuspensions, serum, plasma, liquor), in the case of solid startingmaterials such as tissue, hair roots, blood stains on solid surfaces,cigarette butts, deparaffined tissue etc. additionally by adding waterand carry out the lysis of the starting materials. After completing thelysis of the starting material the lysis batch is incubated in the wayknown as such, if necessary, after adding an ethanolic solution or analcohol/detergent mixture with the various solid phases binding nucleicacid (suspension, centrifugation column) being used. The subsequentbinding of the nucleic acids on the respective solid phases, washing ofthe bound nucleic acids and the final elution will be effected as hasbeen already described according to the state of the art.

[0101] By these solid formulations novel solutions are given, primarilyfor optional fields of application of nucleic acid diagnostics.

[0102] There should be once more pointed out that the invention variantas a one-step method and a “single tube” method allows to isolatenucleic acids from complex starting materials, if necessary, targetamplifications and, if necessary, a subsequent analysing of theamplified nucleic acid section. Thereby, the starting material need notbe a nucleic acid already isolated but the complex starting materialcontaining the nucleic acid. The surface required for binding thenucleic acid contains negative or potentially negative functionalgroups. Binding of the nucleic acid is implemented in a lysis/bindingbuffer with the ions required for binding the negatively charged nucleicacid to the negatively functionalised surface coming from antichaotropicsalts.

[0103] Thus, there are implementable:

[0104] 1. a “single tube” method for isolating nucleic acids fromcomplex starting materials

[0105] 2. a “single tube” method for isolating nucleic acids andsubsequent target multiplication

[0106] 3. a “single tube” method for isolating nucleic acids fromcomplex starting materials, subsequent target multiplication andsubsequent analysing of the multiplied nucleic acid section.

[0107] This means the isolation of nucleic acids from most variousstarting materials containing DNA and if necessary, targetmultiplication and analysing, take place in the very same reactioncavity or, if necessary, on one and the same reaction surface.

[0108] The formulations according to the invention and the universalmethod of binding nucleic acids to solid phases for the isolation,purification and subsequent complex molecular analysis of nucleic acidsfrom optional starting materials and quantities containing nucleic acidsrepresent a novel platform technology for the development of integralfully automated systems of gene analytics which allow to prepare thesample, multiply and analyse the target in one reaction cavity.

[0109] Hereinafter, the invention is explained in greater detail bymeans of examples of execution.

[0110] 1. Isolation of Genomic DNA From Various Plant Materials

[0111] Always 50-100 mg of the starting plant material were comminutedwith a mortar under liquid nitrogen and subsequently transferred to a1.5 ml Eppendorf reaction vessel.

[0112] Adding of 500 μl of lysis buffer (2% CTAB, 2% polyvinylpyrrolidone, 10 mM of tris-HCl, 20 mM of EDTA and 1.3 M of ammoniumchloride) and incubation at 65° C. for at least 30 mm.

[0113] Centrifugating of unlysed components and mixing of thesupernatant liquid with 200 μl of isopropanol.

[0114] Transfer of the solution to a centrifugation column with a glassfiber membrane (micro spin column, company LIDA).

[0115] Centrifugation at 12.000 rpm for 2 min. Discarding of thefiltrate and washing of the membrane two times with a wash buffer (50 mMof NaCl; 10 mM of tris-HCl; 1 mM of EDTA; 70% v/v ethanol).

[0116] After removing ethanol in a short centrifugation step (12,000 rpmfor 2 min.) adding of 200 μl of elution buffer (10 mM of tris-HCl, pH8.7) and elution of DNA by centrifugation at 10,000 rpm for 1 min.

[0117] Always 20 μl of the eluted DNA were put onto an agarose gel andrepresented after coloring with ethidium bromide (FIG. 1).

[0118] 2. Simultaneous Isolation of Genomic DNA From Various StartingMaterials with a Universal Buffer System

[0119] The following samples were used for isolation:

[0120] 1-whole blood frozen: 50 ml, 2-whole blood: 100 μl, 3-cucumber:50 mg; 4-tomato plant leaf: 100 mg; 5-saliva sample: 100 μl; 6-chickenliver; foodstuffs frozen: 5 mg; 7-chicken liver; foodstuffs frozen: 20mg; 8-hair root, 9-turkey salami: 50 mg; 10-yew, needles: 100 mg.

[0121] All samples were incubated in 500 μl of lysis buffer (2% CTAB; 2%polyvinyl pyrrolidone, 10 mM of tris-HCL; 20 mM of EDTA and 1.5 M ofammonium chloride) and with the exception of all plant samples withadding 20 μl of proteinase K (20 mg/ml) at 65° C. Subsequently 200 μl ofisopropanol were added to the lysates and transferred to acentrifugation column with a glass fiber membrane (micro spin column;company LIDA). Centrifugation at 12,000 rpm for 2 min. Discarding of thefiltrate and washing of the membrane twice with a wash buffer (50 mM ofNaCl; 10 mM of tris-HCl; 1 mM of EDTA; 70% v/v ethanol). After removingethanol in a short centrifugation step (12,000 rpm for 2 min.) adding of50-200 μl of elution buffer (10 mM of tris-HCl, pH 8.7) and elution ofDNA by centrifugation at 10,000 rpm for 1 min.

[0122] Always ⅕ of the eluted DNA were loaded onto an agarose gel andrepresented after coloring with ethidium bromide (FIG. 2).

[0123] 3. Isolation of Genomic DNA From Swabs of the Oral Mucosa

[0124] The isolation of DNA from swabs of the oral mucosa is describedhereinafter.

[0125] Always 400 μl of lysis buffer (CTAB, polyvinyl pyrrolidone,ammonium chloride, tris, EDTA) were transferred to a 1.5 ml Eppendorfreaction vessel. The swab material was squeezed out and 20 μl ofproteinase K (20 mg/ml) were added to the suspension. Subsequently thebatch was incubated at 70° C. for 10 min. After lysing 200 μl of adetergent/isopropanol mixture were added, the sample was shaken shortly,subsequently transferred to a commercially available centrifugationcolumn (company LIDA; glass fiber membrane) and centrifuged at 12,000rpm for 1 min.

[0126] Then the column was washed twice with a wash buffer (NaCl,tris-HCl, EDTA, ethanol) containing ethanol (centrifugation at 12,000rpm; 1 min.) and the membrane was dried in a short centrifugation step.By adding 200 μl of elution buffer (10 mM of tris-HCl) the bound DNA waseluted from the filter membrane in a short centrifugation step (10,000rpm; 1 min.) Always 20 μl of the DNA isolated from the two extractionprocesses were placed on 0.7% TAE agarose gel for analysing and analysedafter coloring with ethidium bromide (FIG. 3).

[0127]4. Comparison of the Extraction of DNA From Whole Blood Samples(200 μl) According to the Invention with a Commercial Kit on the Basisof Binding Nucleic Acids in the Presence of Chaotropic Salts

[0128] The isolation of genomic DNA by means of the method according tothe invention was compared with a commercially available andtraditionally applied method for isolating genomic DNA with usingchaotropic salts for binding nucleic acids. The extraction of genomicDNA by means of the comparative method was carried out on the basis ofthe regulation governing application.

[0129] The isolation of DNA by means of the method according to theinvention is described hereinafter.

[0130] Always 200 μl of a whole blood sample (treated with EDTA, fresh)were transferred to a 1.5 ml Eppendorf reaction vessel.

[0131] After adding 350 μl of a lysis buffer (CTAB, polyvinylpyrrolidone, ammonium chloride, tris, EDTA) and 20 μl of proteinase k(20 mg/ml) an incubation was carried out at 70° C. for 10 min. forlysing the starting material.

[0132] After lysing 180 μl of a detergent/isopropanol mixture was added,the sample was shortly shaken, subsequently transferred to acommercially available centrifugation column (company LIDA; glass fibermembrane) and centrifuged at 12,000 rpm for 2 min.

[0133] Then, the column was washed twice with a wash buffer containingethanol (NaCl, tris-HCl, EDTA, ethanol) (centrifugation at 12,000 rpm; 1min.) and the membrane was dried in a short centrifugation step. Byadding 200 μl of elution buffer (10 mM tris-HCl) the bound DNA waseluted from the filter membrane in a short centrifugation step (10,000rpm, 1 min.).

[0134] Always 10 μl of the DNA isolated from the two extractionprocesses were put onto a 0.7% TAE agarose gel and analysed aftercoloring with ethidium bromide.

[0135] The yields of genomic DNA, their integrity (clean individualbands without low-molecular smear bands) and the reproducibility of theextraction methods were compared. As can be seen by means of the methodaccording to the invention better results can be achieved than by meansof the comparative method (FIG. 4).

[0136] 5. Comparison of the Extraction of DNA from Whole Blood Samples(5 μl) According to the Invention by Means of a Commercial Kit on theBasis of Binding Nucleic Acids in the Presence of Chaotropic Salts

[0137] The isolation of genomic DNA by means of the method according tothe invention was compared with the isolation of genomic DNA accordingto a commercially available and traditionally applied method with usingchaotropic salts for binding nucleic acid. Genomic DNA were extractedaccording to the comparative method on the basis of the regulationgoverning application.

[0138] The isolation of DNA by means of the method according to theinvention is described hereinafter.

[0139] Always 5 μl of a whole blood sample (treated with EDTA; fresh)were transferred to a 1.5 ml Eppendorf reaction vessel.

[0140] The sample was filled up to a volume of 200 μl by adding 195 μlof 1×PBS buffer and after adding 350 μl of a lysis buffer (CTAB,polyvinyl pyrrolidone, ammonium chloride, tris, EDTA) and 20 μl ofproteinase K (20 mg/ml) an incubation was carried out at 70° C. for 10min. for lysing the starting material.

[0141] After lysing 180 μl of a detergent/isopropanol mixture wereadded, the sample was shortly shaken, subsequently transferred onto acommercially available centrifugation column (company LIDA, glass fibermembrane) and centrifuged at 12,000 rpm for 2 min.

[0142] Then the column was washed twice with a wash buffer (NaCl,tris-HCl, EDTA, ethanol) (centrifugation at 12,000 rpm; 1 min.) and themembrane was dried in a short centrifugation step. By adding 200 μl ofelution buffer (10 mM of tris-HCl) the bound DNA was eluted from thefilter membrane in a short centrifugation step (10,000 rpm, 1 min.).

[0143] Always 20 μl of the DNA isolated from the two extractionprocesses were put onto a 0.7% TAE agarose gel and analysed aftercoloring with ethidium bromide.

[0144] The possibility of isolating genomic DNA from very smallquantities of starting material and the reproducibility of theextraction processes were detected and compared. As can be seen by meansof the method according to the invention better results can be achievedthan by means of the comparative method (FIG. 5).

[0145] 6. Comparison of the Extraction of DNA According to the InventionFrom Various Types of Animal Tissue Samples and Various Quantities ofStarting Material by Means of a Commercial Kit on the Basis of BindingNucleic Acids in the Presence of Chaotropic Salts

[0146] The isolation of genomic DNA by means of the method according tothe invention was compared with the isolation of genomic DNA accordingto a commercially available and traditionally applied method with usingchaotropic salts for binding nucleic acid. Genomic DNA was extractedaccording to the comparative method on the basis of the regulationgoverning application.

[0147] The isolation of DNA by means of the method according to theinvention is described hereinafter.

[0148] Always 5 mg or 20 mg of tissue samples of pork kidney, pork heartand pork liver were transferred to a 1.5 ml Eppendorf reaction vessel.400 μl of a lysis buffer (CTAB, polyvinyl pyrrolidone, ammoniumchloride, tris, EDTA) and 40 μl of proteinase K (20 mg/ml) were added tothe sample.

[0149] The lysis of the starting material was carried out throughincubation at 52° C.

[0150] After the lysis components not lysed were centrifuged off in ashort centrifugation step (14,000 rpm, 1 min.) and the supernatantliquid was added to a new reaction vessel with 200 μl of adetergent/isopropanol mixture, the sample was shortly shaken,subsequently transferred to a commercially available centrifugationcolumn (company LIDA, glass fiber membrane) and centrifuged at 12,000rpm for 2 min.

[0151] The column was then washed twice with a wash buffer containingethanol (NaCl, tris-HCl, EDTA; ethanol) (centrifugation at 12,000 rpm; 1min.) and the membrane was dried in a short centrifugation step. Byadding 200 μl of elution buffer (10 mM of tris-HCl) the bound DNA waseluted from the filter membrane in a short centrifugation step (10,000rpm., 1 min.).

[0152] Always 10 μl of the DNA isolated from the two extractionprocesses were put onto a 0.7% TAE agarose gel and analysed aftercoloring with ethidium bromide.

[0153] The possibility of isolating genomic DNA from various tissuesamples and various quantities of starting material as regards theyields of genomic DNA , their integrity (clean individual bands withoutlow-molecular smear bands) and the reproducibility of the extractionswere detected and compared.

[0154] As can be seen, by means of the method according to the inventionbetter results can be achieved than by means of the comparative method(FIG. 6).

[0155] 7. Extraction of DNA From Whole Blood Samples (200 μl) by Meansof the Method According to the Invention and Binding of Nucleic Acids toVarious Carriers Used for Binding with Chaotropic Salts Being Mediators

[0156] The isolation of genomic DNA from 200 μl of whole blood by meansof the method according to the invention and binding of the nucleicacids to various carriers (column membranes and suspensions) used forisolating nucleic acids by means of chaotropic agents are represented.

[0157] The extraction of DNA is carried out as described in example 4with various carriers having been used instead of the glass fibermembrane of company LIDA.

[0158] Always 20 μl of the isolated DNA were put onto a 0.7% TAE agarosegel and analysed after coloring with ethidium bromide.

[0159] As is to be seen in FIG. 7, the method according to the inventionimplements the binding of the nucleic acids to various carriers used inthe chaotropic methods which have been known so far.

[0160] 8. Preparation of a Lysis Buffer System Stable in StorageIncluding a Proteolytic Enzyme (Buffer Mix 1) and Use of the LysisBuffer System for Isolating Genomic DNA From Various Starting Materials

[0161] Preparation of a lysis buffer stock solution containing 3 M ofpotassium chloride, 2% CTAB, 18.2 mM of tris-HCl (pH 8.3), 12.5 mM ofEDTA, 2.8% polyvinyl pyrrolidone. Aliquoting of always 400 μl of stocksolution in 1.5 ml Eppendorf reaction vessels and adding of 40 μl ofproteinase K (20 mg/ml).

[0162] Lyophilisation of lysis buffer mixtures in a lyophilisation plant(alpha 2; company Christ). Subsequent storage of the lysis buffermixtures in closed reaction vessels at room temperature for 6 months.

[0163] The extraction of the genomic DNA was carried out from:

[0164] A: 500 μl of whole blood

[0165] B: 400 μl of salivary sample

[0166] C: deparaffined tissue material

[0167] 1. Extraction of DNA from Whole Blood

[0168] Adding of 500 μl of whole blood to the solid formulation of thelysis buffer and incubation at 70° C. for 10 min. Adding of 200 μl ofisopropanol and transfer of the suspension to a centrifugation column(glass fiber mat).

[0169] Centrifugation at maximum speed for 2 min. and discarding of thecentrifugate.

[0170] Adding of 600 μl of a wash buffer (70% ethanol, NaCl, tris,EDTA), centrifugation at maximum speed for 1 min. and discarding of thecentrifugate. Repetition of the washing step. Subsequently drying of themembrane by centrifugation at maximum speed for 2 min. Elution of DNAfrom the membrane by adding 200 μl of an elution buffer (70° C.) andcentrifugation at maximum speed for 1 min.

[0171] 2. Extraction of DNA from Salivary Samples

[0172] Adding of 500 μl of salivary sample to the solid formulation ofthe lysis buffer and incubation at 70° C. for 10 min. Adding of 200 μlof isopropanol and transfer of the suspension to a centrifugation column(glass fiber mat).

[0173] Centrifugation at maximum speed for 2 min. and discarding of thecentrifugate. Adding of 600 μl of a wash buffer (70% ethanol, NaCl,tris, EDTA), centrifugation at maximum speed for 1 min. and discardingof the centrifugate. Repetition of the washing step. Subsequently dryingof the membrane by centrifugation at maximum speed for 2 min.

[0174] Elution of DNA from the membrane by adding 200 μl of an elutionbuffer (70° C.) and centrifugation at maximum speed for 1 min.

[0175] 3. Extraction of DNA from Deparaffined Tissue

[0176] Adding of the deparaffined tissue piece to the solid formulationof the lysis buffer. Adding of 500 μl of dd H₂O and incubation at 52° C.for 30 min.

[0177] Adding of 200 μl of isopropanol and transfer of the suspension toa centrifugation column (glass fiber mat).

[0178] Centrifugation at maximum speed for 2 min. and discarding of thecentrifugate. Adding of 600 μl of a wash buffer (70% ethanol, NaCl,tris, EDTA), centrifugation at maximum speed for 1 min and discarding ofthe centrifugate. Repetition of the washing step. Subsequently drying ofthe membrane by centrifugation at maximum speed for 2 min.

[0179] Elution of DNA from the membrane by adding 200 μl of an elutionbuffer (70° C.) and centrifugation at maximum speed for 1 min.

[0180] Subsequently the extracted DNA was gel electrophoreticallyanalysed. For this purpose, always {fraction (1/10)} of the whole DNAeluate were applied (FIG. 8).

[0181] 9. Preparation of a Lysis Buffer System Stable in StorageIncluding Proteinase K (Buffer Mix 2) and Use of the Lysis Buffer Systemfor Isolating Genomic DNA From 8 Individual Whole Blood Samples (100 μl)

[0182] Preparation of a lysis buffer stock solution containing 3 M ofammonium chloride, 2% polyvinyl pyrrolidone, 16.7 mM of EDTA, 60 mM oftris-HCl, 1.6% CTAB, 20 μl of proteinase K (20 mg/ml).

[0183] Aliquoting of always 400 μl of the stock solution in 1.5 mlEppendorf reaction vessels and incubation of the open Eppendorf reactionvessels in a thermomixer at 95° C. up to its complete drying up.Subsequently closing of the reaction vessels and storage at roomtemperature for 12 months.

[0184] Extraction of DNA from whole blood

[0185] Adding of 100 μl of whole blood to the solid formulation of thelysis buffer and incubation at 70° C. for 10 min. Adding of 20 μl of amineral carrier suspension on silica basis and short mixing. Incubationof the batch for 1 min. Pelleting of the carrier by shortcentrifugation. Washing of the carrier pellets with 800 μl of a washbuffer (70% ethanol, NaCl, tris, EDTA) and subsequent removal of theremaining ethanol by incubation at 70° C. Elution of DNA from thecarrier by adding 200 μl of an elution buffer heated to 70° C. (10 mM oftris-HCl; pH 8.69 and separation of the nucleic acid from the carrier bycentrifugation at maximum speed for 1 min. and transfer of the nucleicacid to a new reaction vessel. The extracted DNA was subsequently gelelectrophoretically analysed. For this purpose, always {fraction (1/10)}of the whole DNA eluate were applied (FIG. 9).

[0186] 10. Isolation of Genomic DNA From Peripheral Blood Lymphocytes bya Direct Binding to the Functionalised Surfaces of a Microtest Plate

[0187] A commercially available plate with a COO⁻ group coating was usedas microtest plate. Always one strip of the plate (8 wells) withfunctional groups and one strip without COO⁻ groups as a negativecontrol were used for the isolation.

[0188] All wells were loaded with 30 μl of peripheral blood lymphocytesin 1×PBS buffer and 180 μl of a lysis buffer (ammonium chloride, CTAB,polyvinyl pyrrolidone, tris-HCl, EDTA, proteinase K) were added andincubated at 70° C. for 5 min. Subsequently 80 μl of adetergent/isopropanol mixture were added. The batches were shortlyshaken and incubated for 5 min. Subsequently the solutions were pouredoff the wells. Thereupon, each of the wells was washed twice with a washbuffer containing ethanol and the remaining ethanol was removed at 70°C. by a short incubation.

[0189] The elution of the nucleic acids was carried out by adding 25 μl10 mM of tris-HCl and an incubation for 2 min.

[0190] Thereupon, the eluates were evaluated on a 0.7% agarose gel (FIG.10).

1. Formulations without chaotropic components for isolating nucleic acids with binding to a solid phase, in particular of DNA, from optional complex starting materials containing a lysis/binding buffer system which contains at least one antichaotropic salt component, a solid phase, wash and elution buffers known as such.
 2. Formulations according to claim 1 wherein the antichaotropic component is an ammonium, caesium, sodium and/or potassium salt, preferably ammonium chloride.
 3. Formulations according to claims 1 or 2 wherein the lysis/binding buffer system contains detergents and additives, if necessary.
 4. Formulations according to claim 3 wherein detergents and additives are tris-HCl, EDTA, polyvinyl pyrrolidone, CTAB, tritonX-100, n-lauryl sarcosine, sodium citrate, DTT, SDS and/or Tween.
 5. Formulations according to claims 1 to 4 wherein the lysis/binding buffer system contains an alcohol for binding to the solid phase.
 6. Formulations according to claims 1 to 5 wherein the lysis/binding buffer system contains enzymes, preferably degrading proteins degrading enzymes.
 7. Formulations according to claims 1 to 6 wherein the lysis/binding buffer system is an aqueous solution.
 8. Formulations according to claims 1 to 6 wherein the lysis/binding buffer system is a solid formulation stable in storage in reaction vessels ready for use.
 9. Formulations according to claims 1 to 8 wherein all carriers serve as a solid phase which were used for isolation by means of chaotropic reagents, preferably glass fiber mats, glass membranes, glasses, zeolites, ceramics, silica carriers.
 10. Formulations according to claims 1 to 8 wherein all carriers which have a negatively functionalised surface or functionalised surfaces which may be converted to a negative charge potential serve as solid phase.
 11. Formulations according to claim 10 wherein the surface of the carrier is modified by an acetyl group, carboxyl group or hydroxyl group.
 12. Method for isolating nucleic acids, in particular DNA, from optional complex starting materials using formulations according to one of the claims 1 to 9 wherein the starting material is lysed, nucleic acids are bound to a solid phase, the nucleic acids bound to the carrier are washed and an elution of the nucleic acids is effected.
 13. Method for isolating nucleic acids according to claim 12 wherein the material containing DNA with a lysisibinding buffer system comprising an aqueous solution which contains an antichaotropic salt component, contains at least a detergent, and if necessary, additives, and if necessary, a proteolytic enzyme and is brought into contact with a solid phase, if necessary, with adding alcohol is washed subsequently and the nucleic acid is dissolved from the solid phase.
 14. Method according to claim 13 wherein starting material are compact plant materials such as fruit; seeds; leaves; needles etc. clinically relevant samples such as whole blood; tissue; microbioptate, paraffine-coated materials, ercp-samples, swabs, foodstuffs such as fish, sausage, tins, milk, forensic samples such as hair roots, cigarette butts, blood stains and other samples containing DNA.
 15. Method according to one of the claims 12 to 14 wherein the ionic strength of the antichaotropic salts for lysis/binding is between 0.1 and 8 M.
 16. Method for isolating nucleic acids, in particular DNA, from optional complex starting materials with using formulations according to one of the claims 1 to 8 and 10 to 11 wherein the starting material is in a “single tube” or one step method chemically modified, brought into contact and lysed with a negatively functionalised surface or its surface in a way that it may be converted to a negative charge potential, the binding of the nucleic acid to the surface is effected, the bound nucleic acid is washed and, if necessary, eluted.
 17. Method according to claim 16 wherein negatively functionalised surfaces are accordingly modified planar surfaces, filter membranes, traditional plastic vessels or microtest plates.
 18. Method according to claims 16 and 17 wherein the nucleic acid is subsequently subjected to an amplification reaction of selected sequence sections in the same reaction batch and, if necessary, thereupon the gene sequences are analysed.
 19. Method according to claims 16 and 17 wherein nucleic acid is subsequently hybridised and/or sequenced in the same reaction batch.
 20. Use of antichaotropic components in a lysis/binding buffer system for isolating and purifying nucleic acids with binding to a solid phase.
 21. Use according to claim 20 wherein antichaotropic components are ammonium, caesium, sodium and/or potassium salts, preferably ammonium chloride.
 22. Use according to claims 20 or 21 wherein antichaotropic salts in an ionic strength between 0.1 and 8 M are used for lysis/binding.
 23. Use according to one of the claims 20 to 22 wherein the lysisibinding buffer system is used as an aqueous solution.
 24. Use according to one of the claims 20 to 22 wherein the lysisibinding buffer system is present as a stable formulation stable in storage.
 25. Use according to one of the claims 20 to 24 for the preparative isolation and purification of DNA for use in gene therapy. 